| Part ? Synthesis,characterization of FHA and SiHA nanoparticlesObjective:The aim of this study is to present the synthesis process of HA,FHA,and SiHA nanoparticles and detect the physical and chemical characteristics of materials.Materials and methods:HA,FHA and SiHA nanoparticles were prepared by Aqueous Precipitation Method respectively.Ca(NO3)2 was used as the calcium source,(NH4)2HPO4 as the phosphorus source,NH4F as the fluorine source,and Tetraethoxysilane(TESO)as the silicon source.SEM was used to detect the surface morphology of the material,and TEM was used to detect the internal morphology of the material.XRD test detected internal pores and lattice structures.XPS analysis confirmed the chemical composition of HA,FHA and SiHA nanoparticles.Results:SEM and TEM showed that HA,FHA and SiHA nanoparticles were all rod-shaped.HA was 200nm at length and 20nm at diameter.FHA was 800nm at length and 200nm at diameter.SiHA is about 500 nm at length and 50 nm at diameter.XRD analysis showed the HA in the experiments was standard HA.With the modification of F,the crystallinity increases compare to pure HA.After silicon is introduced,the crystallinity decreases compare to HA.XPS results showed that Ca,O,and P are the constituent elements of HA,and the ration of Ca:P=1.66;Ca,O,P,F are the constituent elements of FHA,and the ratio of F:OH=1.1:0.9;and SiHA was contributed with element of Ca,O,P and Si,and the wt%of Si was 0.77wt%.Conclusion:FHA and SiHA nanoparticles were successfully synthesized.Part ? The comparison study of osteogenic induction properties of FHA and SiHA nanoparticles in vitro and in vivoExperiment ? The comparison study of osteogenic induction properties FHA and SiHA nanoparticles in vitroObjective:The aim of this part is to extract and identify BMSCs.The biocompatibility of HA,FHA and SiHA nanoparticles were also detected.Materials and methods:The Bone Marrow Mesenchyml Stem Cells(BMSCs)were extracted from SD rats by flushing bone marrow out of femur and tibia.Alizarine Red stanning,Oil Red stanning and Alcian blue staining were taken to detect osteogenic,adipogenic,and chondrogenic differentiation of BMSCs.Flow cytometry was used to measure the protein expression of CD44,CD45,CD11b and CD29 on the cell membrane.MTT assay was used to detect the proliferation of BMSCs in HA,FHA and SiHA nanoparticle extracts.F ion analyzers were used to determine the F-release concentration of FHA.Plasma emission spectra used to determine the SiO44concentration of SiHA.BMSCs were cultured with HA,FHA and SiHA nanoparticles extracts with concentration of 10?g/mL,2.5?g/mL and 0.625?g/mL,and MTT method was used to determine the best concentration.BMSCs were cultured 1,4 and 7 days,and cellular fluorescence staining was used to detect BMSCs morphology and adhesion when cultured in HA,FHA and SiHA nanoparticles extracts.The osteogenic activity level of BMSCs was analyzed by Alizarin Red staining and ALP activity.The mRNA expression of Runt-related Transcription Factor 2(Runx2),Osteocalcin(Ocn),Osteopontin(Opn)were detected by RT-PCR analysis.Results:BMSCs of SD rats showed its capacity of osteogenic,adipogenic and chondrogenic differentiation.The positive expression of CD44 and CD29 is more than 80%and the negative expression of CD45 and CDllb is less than 10%according the result of flow cytometry.MTT test showed that HA,FHA and SiHA nanoparticles extraction has no cytotoxicity at a concentration of 0.625?g/mL and all the three nanomaterial extracts can promote cell proliferation.BMSCs in the HA and SiHA nanoparticles extraction were in good morphology.However,cell started to shrink in the FHA extracts from 7th day.After 14 days of osteogenic induction,BMSCs cultured in FHA nanoparticles extraction had formed more and bigger mineralized nodules compare to that in HA and SiHA nanoparticles extraction.The mRNA expression of Runx2,Opn and Ocn of cells cultured in FHA nanoparticles extraction were significantly higher than that in HA and SiHA nanoparticles extraction.Conclusion:BMSCs of SD rats were successfully derived.BMSCs in SiHA group exhibited relatively lower viability than in HA and FHA groups.The ability to promote osteogenesis in vitro can be ranked as following:FHA>SiHA?HA>blank group.Experiment ? The comparison study of osteogenic induction properties of FHA and SiHA nanoparticles in vivoObjective:The aim of the study was to evaluate the osteogenic potential and the influence on osteoclasts of HA,FHA and SiHA nanoparticles in the femoral metaphyseal defect model of SD rats.Materials and methods:Eight-week-old female SD rats were used to establish bilateral femoral metaphyseal bone defect models and implanted with HA,FHA,and SiHA nanoparticle materials,respectively.The therapeutic efficiency of HA,FHA and SiHA nanoparticles were evaluated by ?CT and analysis the data of BV/TV,Tb.Th,BS/BV,Tb.Sp and Tb.N.H&E staining in rat femur defect was used to evaluated the new bone regeneration.TRAP staining in rat femur defect was used to evaluated the osteoclast around bone defect.Results:?CT and H&E staining elucidated significant discrepancy of new bone formation at 4weeks than 8 weeks in all groups.SiHA presented the best bone repair effect especially at 8 weeks.However,TRAP staining showed the osteoclasts in SiHA group exhibited the most compared to the other groups.Conclusion:The ability to promote osteogenesis in vivo can be ranked as follows:SiHA>FHA?HA>blank group.Part ? The comparison study on antibacterial ability to Porphyromonas gingivalis of FHA and SiHA nanoparticlesObjective:The aim of the study was to assess the differences of HA,FHA and SiHA nanoparticles in antibacterial performance against Porphyromonas gingivalis(P.gingivalis).Materials and method:Round tablets of HA,FHA and SiHA nanoparticle materials are co-cultured with P.gingivalis.The antibacterial properties of the three materials on P.gingivalis were evaluated through plate counting and live-dead bacterial staining.Results:After bacteria was cultured with HA,FHA,and SiHA tablets,the monoclonal colony counting of P.gingivalis showed that the number of bacteria cultured with FHA counts was less than with SiHA and HA tablets.The amount of both live and dead bacteria on the HA and SiHA tablets were more than that on FHA tablet.Conclusion:The FHA nanoparticle exhibited the highest antibacterial activity against P.gingivalis.Part ? Effect of FHA nanoparticles on Expression of Inflammatory Cytokines and Osteogenic Differentiation of MC3T3-E1 Stimulated by LPSObjective:An inflammatory model of MC3T3-E1 in vitro was established by P.gingivalis Lipopolysaccharide(LPS).The study of this part was to investigate the effect of FHA nanoparticles on the inflammatory model of MC3T3-E1 in vitro.Materials and method:The concentration of 1?/mL,5?g/mL and 10?g/mL concentrations of P.gingivalis LPS stimulated MC3T3-E1 for 6,12,24h respectively,and the mRNA expression levels of IL-1?,IL-6,TNF-? were measured by RT-PCR to find out the best concentration and time to build the inflammatory model of MC3T3-E1.The cytotoxicity of optimal concentrations of P.gingivalis LPS on MC3T3-E1 was detected by CCK8 assay.RT-PCR was used to investigated the mRNA levels of IL-1?,IL-6 and TNF-? in model of MC3T3-E1 inflammation in vitro.RT-PCR was used to investigated the mRNA expression of Alp,Runx2,Ocn,Opn,Vegf and Osx after culturing with osteogenic induction 7,14 and 21 days.Alizarin Red staining and ALP activity were used to analyze the effects of FHA nanoparticles extracts on the osteogenic activity.Result:RT-PCR results showed that MC3T3-E1 stimulated by 5?g/mL P.g LPS for 12 h could significantly induce the mRNA expression of IL-6,IL-1? and TNF-?,and the concentration with 5?g/mL of P.gingivalis LPS had no toxicity to the MC3T3-E1.Low concentration(1?g/mL)of FHA nanoparticles extract significantly reduced the mRNA expression levels of inflammatory cytokines such as IL-6,IL-1? and TNF-?.While high concentration of FHA nanomaterial extract(5g/mL)stimulated the inflammatory model of MC3T3-E1 for 24 hours could increase the expression of inflammatory factors.After continuous osteogenic induction for 7,14 and 21 days,1?g/mL FHA nanomaterial extract could significantly increase the mRNA expression level of osteogenic related genes,but lower than that in normal cells.Alizarin red staining and ALP staining showed the similar results with RT-PCR.Conclusion:The model of osteogenic inflammation in vitro was successfully established by stimulating the MC3T3-E1 with 5?g/mL P.gingivalis LPS for 12h.Low concentration(1?g/mL)of FHA nanoparticles extract can reduce the mRNA expression levels of IL-6,IL-1? and TNF-? in MC3T3-E1 inflammatory model in vitro.1 ?g/mL FHA nanoparticle extraction might promote the bone formation by inhibiting the expression of inflammatory cytokines in MC3T3-E1 irritated by P.gingivalis LPS. |
PDF Full Text Request